Perinatal hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal death and long-term disability. To date, no individual neuroprotective agent has been proven safe and effective, although clinical trials have shown benefits of hypothermia in improving outcomes in HIE infants. Novel therapies optimized for this devastating disease are urgently needed. Clinically, males are more sensitive to neonatal HIE; however, the mechanisms underlying the sex difference are unknown. The innate immune response has a fundamental role in the pathophysiology of HIE. Microglial activation is the key initiator of the immune response, and is regulated by the endogenous inhibitory signals, primarily CX3CL1/CX3CR1 signaling pathway. Recent studies have found that sexual dimorphism exists in microglia number, activation, and expressed membrane receptors in the neonatal brain under normal conditions. Nevertheless, how these basal sex differences affect the response to a neonatal injury such as HIE remains largely unexplored. We hypothesize that microglia are differentially activated after HIE in male and female neonates, leading to differential immune responses and ischemic outcomes. The Rice-Vannucci model will be used to induce HIE in post-natal day 10 (P10) mice of both sexes. In Aim 1, we will test the hypothesis that sex differences exist in microglial activation and in HIE outcomes due to the sexual dimorphism in CX3CL1/CX3CR1 signaling. Pharmacological enhancement and genetic deletion of CX3CL1/CX3CR1 signaling pathway will be performed to mechanistically study the effect of manipulation of this pathway on HIE. In Aim 2, we will use Christmas mouse (CX3CR1gfp/+CCR2rfp/+) to investigate the sexual dimorphism in central and peripheral immune response to HIE. The Christmas mouse model allows us to differentiate blood-derived macrophages from resident microglia. By using Christmas mice together with flow cytometry and IHC, we will be able to study the sex difference in both the central and peripheral immune response, and investigate the role of each component (central vs. peripheral) in HIE. These exploratory studies will lead to better understanding of sex-specific mechanisms underlying HIE and will help us identify and optimize biological targets for therapeutic intervention in children.